Development and application of specific FISH probes for karyotyping Psathyrostachys huashanica chromosomes

Background Psathyrostachys huashanica Keng has long been used as a genetic resource for improving wheat cultivar because of its genes mediating the resistance to various diseases (stripe rust, leaf rust, take-all, and powdery mildew) as well as its desirable agronomic traits. However, a high-resolution fluorescence in situ hybridization (FISH) karyotype of P. huashanica remains unavailable. Results To develop chromosome-specific FISH markers for P. huashanica, repetitive sequences, including pSc119.2, pTa535, pTa713, pAs1, (AAC)5, (CTT)12, pSc200, pTa71A-2, and Oligo-44 were used for a FISH analysis. The results indicated that the combination of pSc200, pTa71A-2 and Oligo-44 probes can clearly identify all Ns genomic chromosomes in the two P. huashanica germplasms. The homoeologous relationships between individual P. huashanica chromosomes and common wheat chromosomes were clarified by FISH painting. Marker validation analyses revealed that the combination of pSc200, pTa71A-2, and Oligo-44 for a FISH analysis can distinguish the P. huashanica Ns-genome chromosomes from wheat chromosomes, as well as all chromosomes (except 4Ns) from the chromosomes of diploid wheat relatives carrying St, E, V, I, P and R genomes. Additionally, the probes were applicable for discriminating between the P. huashanica Ns-genome chromosomes in all homologous groups and the corresponding chromosomes in Psathyrostachys juncea and most Leymus species containing the Ns genome. Furthermore, six wheat–P. huashanica chromosome addition lines (i.e., 2Ns, 3Ns, 4Ns, 7Ns chromosomes and chromosomal segments) were characterized using the newly developed FISH markers. Thus, these probes can rapidly and precisely detect P. huashanica alien chromosomes in the wheat background. Conclusions The FISH karyotype established in this study lays a solid foundation for the efficient identification of P. huashanica chromosomes in wheat genetic improvement programs.

has many agronomically valuable characteristics, including early maturation, the production of multiple kernels and tillers, drought tolerance, and resistance to biotic factors (e.g., pathogens causing stripe rust, leaf rust, takeall, and powdery mildew) [3]. Moreover, it has been used as a tertiary gene resource for wheat genetic improvement ever since chromosome-mediated gene transfers from P. huashanica into common wheat were initiated in the 1990s [2]. Progeny lines carrying P. huashanica chromosomes or chromosomal segments incorporated into the wheat genome were subsequently developed as lines with chromosomal additions [4][5][6], substitutions [7,8], and translocations [9,10]. These lines outperformed their wheat parents in terms of stress resistance and agronomic traits, making them valuable germplasm for wheat breeding. Molecular markers, including randomamplified polymorphic DNA markers targeting the P. huashanica Ns genome [11] and sequence characterized amplified region markers specific for the 1Ns, 2Ns, 3Ns, and 5Ns chromosomes [4,[12][13][14], have been developed for exogenous chromatin tracking.
Fluorescence in situ hybridization (FISH) is a widely used cytogenetic tool for detecting chromosomal aberrations as well as for studying chromosomal behavior and the genomic localization of repetitive DNA sequences [15,16]. The FISH karyotype established on the basis of informative probe labeling patterns on chromosomes may provide researchers with landmarks useful for identifying chromosomes. In previous FISH studies, repetitive sequence probes, such as pSc119.2, pTa535, pTa713, pAs1, and microsatellites like (AAC) 5 , enabled the convenient identification of wheat chromosomes according to distinct probe hybridization patterns [17][18][19][20]. Such FISH karyotye has also been conducted in investigations of the diploid Thinopyrun elongatum [21], Dasypyrum villosum [22], Agropyron cristatum [23], Secale cereale [24], Hordeum vulgare [25], and Aegilops species, including Ae. markgrafii, Ae. cylindrica, Ae. triuncialis [26], Ae. comosa, Ae. geniculata, Ae. biuncialis and Ae. umbellulata [27,28]. To date, few repetitive sequences have been used as FISH probes for analyzing P. huashanica chromosomes. Furthermore, a detailed FISH karyotype based on homoeologous relationships with wheat is not available for the P. huashanica Ns-genome chromosomes. A lack of a standard P. huashanica karyotype has greatly hindered the chromosome-mediated transfer of elite genes from P. huashanica into cultivated wheat.
Although FISH using repetitive sequence probes has been widely used for identifying chromosomes in numerous Triticeae species [19][20][21][22][23][24][25][26][27][28][29], it is not a viable option for determining inter-genera chromosomal homoeologous relationships because of the broad variation in these repetitive elements among diverse genomes [30,31]. Chromosome painting using chromosome-specific probes, which are designed on the basis of single-copy sequences, is a novel cytological method for diagnosing chromosomal abnormalities, detecting chromosomal rearrangements, investigating chromatin organization, and constructing ancestral karyotypes [32]. Li et al. [33] recently developed seven oligonucleotide pools (Synt1-Synt7) derived from the conserved sequences in the collinear chromosomal regions of each linkage group in wheat and barley. These pools were used to differentiate between the chromosomal homeologous groups in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum. Accordingly, these newly synthesized linkage group-specific bulked oligonucleotide pools are applicable for investigating chromosomal homoeologous relationships among Triticeae species.
In our previous studies, we generated hybrids from a cross between P. huashanica and common wheat without an embryo rescue step and obtained hybrid derivatives by backcrossing and selfing [34]. A detailed FISH karyotype of P. huashanica chromosomes remains unavailable and there are relatively few P. huashanica-specific molecular markers. This has made it difficult to identify wheat-P. huashanica progenies useful for breeding. To optimize the utility of P. huashanica desirable genes for the genetic improvement of wheat via chromosome engineering, FISH-based markers that can efficiently identify P. huashanica chromosomes in the wheat background are urgently needed. Thus, the objectives of this study were to: (1) develop a FISH-based karyotype specific for the P. huashanica Ns-genome chromosomes; (2) validate the P. huashanica chromosome-specific FISH markers in common wheat and wheat-related species; and (3) identify wheat-P. huashanica derivative lines using the developed FISH markers.

Development of P. huashanica chromosome-specific probes
Actively growing root tips from germinating seeds or growing plants were cut and treated with nitrous oxide for 2.5 h and 70% (v/v) acetic acid for 5 min before being digested with pectinase and cellulase (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) [19]. Root tips for outcrossing species were taken from randomly selected individual plants or seed of each accession. The FISH and GISH slides were prepared as described by Han et al. [38]. To construct the P. huashanica karyotype, the pSc200, pTa71A-2, and Oligo-44 repetitive sequences were selected for the FISH experiments after screening other oligonucleotides probes, including pSc119.2, pTa535, pTa713, pAs1, (CTT) 12 , and (AAC) 5 . Sequence of each probe was listed in Table 3 and all probes were 5' end-labelled with 6-carboxyfluorescein or 6-carboxytetramethylrhodamine by Sangon (Shanghai, China). The hybridization was completed according to a slightly modified version of the method described by Han et al. [38]. Briefly, 10 µL hybridization solution comprising the probes and buffer [0.5 µL each probe in 2× saline sodium citrate (SSC) and 1× TE buffer] was added to each slide. The samples were denatured at 80 °C for 5 min and then incubated at 37 °C for 2 h, after which they were washed with 2× SSC buffer. The chromosomes were counterstained with a DAPI (4′,6-diamidino-2-phenylindole) solution (Vector Laboratories, Inc., Burlingame, CA,  USA) before photomicrographs of the chromosomes were taken using the DP80 CCD camera (Olympus, Tokyo, Japan) installed on the BX-63 microscope (Olympus). The photomicrographs were processed using Photoshop CS5.0 (Adobe Systems Incorporated, San Jose, CA, USA).

Karyotyping of P. huashanica chromosomes
To distinguish the P. huashanica homoeologous chromosomes from the wheat chromosomes, bulked oligonucleotide libraries (Synt1-Synt7) kindly provided by Prof. Z.J. Yang (Center for Informational Biology, School of Life Science and Technology, University of Electronic and Technology of China) were used for FISH painting. The washed slides were used for a FISH involving the oligonucleotide probes pTa71A-2 and Oligo-44. The FISH painting was performed as described by Han et al. [44] and Bi et al. [45]. Slides were washed as described by Komuro et al. [19]. Photomicrographs were taken as described in the FISH protocol. Fifteen well-spread metaphase cells of P. huashanica accession ZY3157 were selected for an analysis of chromosomal parameters. The relative length was calculated by dividing the length of a particular chromosome by the total length of chromosomes in the haploid set. The chromosomal arm ratio was determined by dividing the length of the longer arms by the length of the shorter arms. Data were processed using the Image J software and Microsoft Office Excel 2010.

Validation of the P. huashanica-specific FISH-based markers
To validate the specificity and utility of the developed cytological markers, pSc200, pTa71A-2, and Oligo-44 were used for a FISH analysis of CS, 13 wheat relatives, and wheat-P. huashanica derivatives. A GISH was performed using the washed FISH slides for the derived lines to confirm the presence of P. huashanica chromatin. For both P. huashanica and CS, genomic DNA was extracted from fresh leaves according to the cetyltrimethylammonium bromide method [46]. The P. huashanica (ZY3157) genomic DNA was labeled with Texas Red-12-dUTP in the nick translation mix (Thermo Fisher Scientific, Eugene, OR, USA) and then used as the GISH probe. The CS DNA was used for blocking. The GISH was performed according to a modified version of the method described by Han et al. [38]. Specifically, 10 µL GISH hybridization solution containing 100 ng labeled probe DNA and blocking DNA (1:150 probe DNA:blocking DNA ratio), 2× SSC, 10% (v/v) dextran sulfate, and 50% (v/v) formamide was added to each slide. The samples were denatured at 85 °C for 5 min, incubated at 50 °C overnight, and washed with 2× SSC buffer. The chromosomes were counterstained and photomicrographs were taken as described in the FISH protocol.

Karyotyping of P. huashanica Ns-genome chromosomes
Several DNA probes, including pSc119.2, pTa535, pTa713, pAs1, (AAC) 5 , (CTT) 12 , pSc200, pTa71A-2, and Oligo-44, were used for the FISH analysis of P. huashanica chromosomes (Figs. 1 and 2B C). The FISH results indicated that the pTa71A-2 and Oligo-44 probe combination unambiguously discriminated the P. huashanica chromosomes (Fig. 1 F). The probes pTa71A-2 and Oligo-44, which were associated with pSc200, enabled the precise identification of all P. huashanica Ns-genome chromosomes (Fig. 2B C). By combining the oligonucleotide probes for FISH images and the bulked pool probes (Synt1-Synt7) for the seven Triticeae linkage groups (Fig. 3), the FISH patterns of P. huashanica chromosomes 1Ns to 7Ns were established (Fig. 2 A). Details regarding chromosome morphology are summarized in Table 4.  TTT TGT GGA CTA TTA CTC ACC GCT TTG GGG TCC CAT AGC TAT  [17] pTa535 AAA AAC TTG ACG CAC GTC ACG TAC AAA TTG GAC AAA CTC TTT CGG AGT ATC AGG GTTTC [17] pAs1 CCT TTC TGA CTT CAT TTG TTA TTT TTC ATG CAT TTA CTA ATT ATT TTG AGC TAT AAGAC [17] (AAC) 5 (AAC) 5 [20] pTa713 AGA CGA GCA CGT GAC ACC ATT CCC ACC CTG TCT TAG CG [39] (CTT) 12 (CTT) 12 [40] pTa71A-2 CCG ACG GCC GTC GTG GAC GGA AGT TGA CGC GCG CCA TGG AAA ACTG [41] Oliog-44 TAG CTC TAC AAG CTA GTT CAA ATA ATT TTA CAC TAG AGT TGA AC [42] pSc200 CTC ACT TGC TTT GAG AGT CTC GAT CAA TTC GGA CTC TAG GTT GAT TTT TGT ATT TTCT [43] Synt1-Synt7 \ [33] The chromosomes of the two P. huashanica accessions had similar probe hybridization patterns, with some variations in the FISH signals for the 1Ns homologous chromosomes analyzed using probes pTa71A-2 and pSc200, and for 2Ns analyzed using probe pSc200, which hybridizes exclusively to the terminal region of all chromosomes ( Fig. 2 A). Chromosome 1Ns was characterized by heteromorphic pTa71A-2 signals, with varying densities at the nucleolar organizing regions (NORs) of the short arms among the homologous chromosomes. Additionally, strong Oligo-44 signals were detected near the centromere region of the long arms. Terminal pSc200 signals were observed at the short arms of chromosome 1Ns. Moreover, pSc200 revealed the heteromorphism at the terminal region of the long arms. Chromosome 2Ns had clear pTa71A-2 signals at the subterminal region of the long arms. The variation at the terminal region of the 2Ns chromosomes detected using pSc200 was identical to that on the 1Ns chromosomes. Chromosome 3Ns was identified on the basis of weak Oligo-44 signals in pericentromeric and strong Oligo-44 signals near the centromere region of the long arms; strong terminal pSc200 signals were detected on both arms. Chromosome 4Ns lacked specific pTa71A-2 and Oligo-44 signals, but strong terminal pSc200 signals were observed on both arms. Clear pTa71A-2 and faint pSc200 signals were simultaneously produced at the terminal of the short arms of 5Ns chromosomes, with strong Oligo-44 signals at the intercalary region of the long arms. Chromosome 6Ns had strong Oligo-44 signals near the centromere region of the long arms and clear terminal pSc200 signals on both arms. Notably, distinct Oligo-44 signals were observed at the middle region of both arms of chromosome 7Ns. Terminal pSc200 signals on chromosome 7Ns were the same as those on chromosomes 3Ns, 4Ns, and 6Ns (Fig. 2 A).

Validation of the FISH markers
To validate the specificity of the FISH markers for P. huashanica Ns-genome chromosomes, probes pTa71A-2, Oligo-44, and pSc200 were used to perform a FISH analysis of the metaphase cells of CS and six diploid wheat relatives containing St, E, V, I, P and R genomes, respectively (Table 1). There was a lack of pSc200 signals on all the chromosomes of wheat, Pse. revealed polymorphic hybridization sites on several chromosomes in all species. Combining pSc200 with Oligo-44 or pTa71A-2 enabled the discrimination of all P. huashanica Ns-genome chromosomes, with the exception of the 4Ns chromosomes, from the P-and R-genome chromosomes, some of which had terminal pSc200 signals that were the same as those on the 4Ns chromosomes (Fig. 4). These probes were also used for a FISH analysis of seven Ns-genome-containing species to assess their utility for distinguishing between P. huashanica chromosomes and other Ns-genome chromosomes in a different genetic background. The pSc200 signals were mainly detected at the terminal region of one or both arms of the Ns-genome chromosomes in most of the examined species. The exceptions were P. juncea, in which faint signals were detected primarily at the subterminal region of several chromosomes, and L. arenarius, in which signals were undetectable. The pSc200 signal distribution patterns were not specific to particular chromosomes or genome types, but the strong terminal signals on all P. huashanica chromosomes except chromosome 5Ns were useful for distinguishing from the Ns j , Ns s , Ns p , Ns m , and Ns a chromosomes to some extent. All of the identified Ns-genome chromosomes in the different species had Oligo-44 FISH patterns that were similar to those of P. huashanica chromosomes belonging to homologous groups 1, 3, 5, 6, and 7, but there were obvious interspecific differences in the signal localization for the 1Ns 1 a and 1Ns 2 a chromosomes, all 5Ns chromosomes (except for 5Ns m and 5Ns c ), chromosome pairs 6Ns s , 6Ns p , 7Ns j , 7Ns p , 7Ns m , 7Ns 2 a , and a single 7Ns s and 7Ns c chromosome. The mapping of the pTa71A-2 sites on these Ns-genome chromosomes revealed different 45 S rDNA loci that were the same or similar in size, which provided limited information useful for discriminating between chromosomes (Fig. 5).
Using the three probes simultaneously for a FISH analysis unambiguously distinguished chromosome 1Ns h from the homologous chromosomes in all other species. Chromosome 2Ns h could also be distinguished from all other 2Ns chromosomes by the pSc200 and pTa71A-2 probe combination. The pSc200 and Oligo-44 probes were able to differentiate chromosome 3Ns h from the other 3Ns chromosomes. Probes pTa71A-2 and Oligo-44 failed to hybridize to chromosome 4Ns h , whereas pSc200 was able to discriminate this  A, B, C, D, E, F, G, H) FISH painting using bulked oligo probes Synt1 (red) + Synt2 (green), Synt3 (red) + Synt4 (green), Synt5 (green) + Synt6 (red) and Synt7 (green) with corresponding FISH using probes pTa71A-2(green) + Oligo-44(red). Scale bar: 10 μm and Ns c ) in homologous groups 6 and 7 could also be clearly distinguished using the pSc200 and Oligo-44 probe combination (Fig. 5).

Utility of the FISH markers for identifying P. huashanica chromosomes in the wheat background
To evaluate the potential utility of pTa71A-2, Oligo-44, and pSc200 for identifying P. huashanica chromosomes in the wheat background, a FISH was performed on the metaphase cells of wheat-P. huashanica hybrid derivatives. Six wheat-P. huashanica chromosome addition  (Fig. 6 A,  6B). Line 32-2-5 (2n = 43) was identified as a monosomic chromosome addition line containing a 3Ns chromosome on the basis of the signal patterns and a larger arm ratio than that of the 6Ns chromosomes (Fig. 6C, D). The probe signals indicated that line 26-3-2 (2n = 44) carried an additional 2Ns and 4Ns chromosome respectively (Fig. 6E F), whereas line 18-2-6 (2n = 44) was a disomic chromosome addition line with a pair of 7Ns chromosomes (Fig. 6G H). Line 20-1-1 (2n = 44) was a 2NsL ditelosomic addition line. Although line 23-1-1 (2n = 44) was also identified as a ditelosomic addition line, it was unclear which group the additional chromosomes belonged to (Fig. 6I L).

Discussion
The ability to accurately identify alien chromosomes in the wheat background is critical for exploiting alien genetic resources [39]. Accordingly, FISH analyses involving pSc119.2, pTa535, pTa713, pTa794, pAs1, and the microsatellites (AAC) 5 and (CTT) 12 have been widely conducted to identify chromosomes from wheat [17], S. cereale [47], diploid Th. elongatum [21], Das. villosum [22], and Aegilops species (S-, U-, C-, N-, D-and M-genomes) [26-28, 32, 48, 49]. However, the resolution of the results obtained by combining these frequently used FISH probes was insufficient for discriminating the seven pairs of P. huashanica chromosomes (Fig. 1). By conducting a FISH analysis using repetitive sequences, we revealed that all P. huashanica Ns-genome chromosomes can be unambiguously discriminated from each other by pTa71A-2 and Oligo-44 ( Fig. 1 F). Moreover, the detailed FISH karyotype constructed using pSc200, pTa71A-2, and Oligo-44 enabled the accurate identification of all P. huashanica chromosomes (Fig. 2). Badaeva et al. [50] developed the FISH karyotypes of 12 diploid Aegilops species and divided the chromosomes into homoeologous groups according to the standard C-banding patterns. Zhang et al. [51] constructed a FISH-based map of H. villosa chromosomes on the basis of the FISH patterns on the V-genome chromosomes from a whole set of wheat-H. villosa disomic chromosome addition lines. Said et al. [23] arranged the P-genome chromosomes by mapping conserved single-copy wheat cDNAs on the homoeologous chromosomes of A. cristatum.
Using linkage group-specific bulked oligonucleotide pools, we constructed the P. huashanica karyotype relatively simply and quickly. Notably, FISH using pSc200, Oligo-44, and pTa71A-2 together distinguished the P. huashanica Ns-genome chromosomes from the wheat chromosomes as well as all chromosomes except for 4Ns from the chromosomes of diploid wheat relatives containing St, E V, I, P and R genomes, while also differentiating between the P. huashanica Ns-genome chromosomes in all homologous groups and those of P. juncea and most Leymus species. A previous study proved that for blotting and FISH analyses, pSc200 from rye telomeric/subtelomeric repetitive sequences does not produce detectable signals in the wheat genome [52]. Hence, it was used to differentiate the R-genome chromosomes of rye from the A-, B-, and D-genome chromosomes of wheat [43]. It has also been used to detect changes in the telomeric/subtelomeric sequences of rye chromosomes in wheat-rye amphidiploids [53]. In this study, the terminal pSc200 signals on all P. huashanica chromosomes enabled the discrimination of the P. huashanica chromosomes from the wheat chromosomes and revealed the variation of telomeric sequences from the Ns-genome chromosomes in P. juncea and Leymus species (Figs. 2 and 4 C, and 5). Probe pTa71A-2, which belongs to the pTa71 family of repeating sequences (45 S rDNA), comprises a 9-kb sequence from the common wheat 18 S-5.8 S-26 S rDNA and intergenic spacers [41,54]. The 45 S rDNA gene locus is usually linked with NORs in the tribe Triticeae. Previous research indicated that major NORs are preferentially located on the short arms of chromosomes in homoeologous groups 1, 5, and 6 [54,55]. However, according to the results of this study, pTa71A-2 hybridizes to the short arms of the 1Ns and 5Ns chromosomes while the long arms of the 2Ns chromosomes (Fig. 2 A). The positions of major NORs determined using pTa71A-2 in this study were in accordance with the findings of an earlier study by Kisimi [56]. Thus, the differences in the major 45 S rDNA sites on the long arms of the 2Ns chromosomes in P. huashanica were likely due to a chromosomal non-homologous recombination between 6Ns and 2Ns. Li et al. [33] confirmed the non-homologous chromosomal rearrangements in rye, Ae. uniaristata, Ae. markgrafii, and Ae. umbellulata using linkage group-specific bulked oligo pools. In contrast, we did not detect interchanges involving P. huashanica 2Ns and 6Ns chromosomes because the probe pools mainly hybridized to the non-terminal region of both arms of the P. huashanica chromosomes (Fig. 3 A, C, E, and G), indicative of a relatively low homology between the telomeric regions of wheat and P. huashanica chromosomes. Oligo-44 is a new tandem repeat isolated from the CS genome that has been used to identify specific wheat chromosomal segments [42]. In the current study, abundant Oligo-44 hybridization signals facilitated the identification of P. huashanica chromosomes, and they also helped the detection of several Ns-genome chromosomes in P. juncea and Leymus species (Fig. 5), which contain the Ns genome from the genus Psathyrostachys and the Xm genome from unconfirmed donors [57]. In addition to the pSc200 and pTa71A-2 signals, polymorphic Oligo-44 hybridization patterns elucidated the genetic differences among the Ns-genome chromosomes in diverse genetic backgrounds (Fig. 5), which were previously determined in Southern blots and via molecular studies [58][59][60].
The distribution of Oligo-44 signals provided some insights into the relationship between Ns and Xm genomes. On the basis of the chromosome pairing in intergeneric hybrids and the results of DNA hybridization experiments, the evolutionary distance between the Ns and Xm genomes in Leymus species has been investigated. In earlier studies by Dewey [61] and Wang et al. [62], the trivalent ratio was low during analyses of the configuration of F 1 pollen mother cells at metaphase I in Leymus × Psathyrostachys hybrids, suggesting the tetraploid Leymus species are allotetraploids. Zhang et al. [63] subsequently confirmed their findings using GISH and chromosome pairing data. However, FISH analyses involving the mapping of dispersed retrotransposon-like repeats from L. mollis and L. arenarius on the chromosomes of Psathyrostachys and Leymus species indicated that Leymus species should be considered as autopolyploids with the Ns genome or segmental allopolyploids consisting of an altered basic Ns genome [64]. The Oligo-44 signal distribution characteristics among the Ns-genome chromosomes indicated that the corresponding repetitive sequence was conserved during the evolution of the Ns genome ( Fig. 5; Table 5). Thus, there are theoretically 2-times and 4-times more chromosomes carrying Oligo-44 signals of assumed autotetraploid and autooctaploid Leymus species, respectively, than in diploid Ns-genome-containing species. In the current study, the number of chromosomes giving Oligo-44 hybridization sites was 10-13 in tetraploid Leymus species and 22 in the octaploid L. arenarius (Table 5), implying that Leymus species are likely not autopolyploids, which is consistent with the available DNA sequence information [65,66]. Because small-scale analyses may lead to incomplete conclusions, more comprehensive approaches are required for future biosystematics-based investigations of the genus Leymus.
The drawbacks to the conventional methods for detecting P. huashanica chromosomes (e.g., analyses involving Giemsa C-banding technology or molecular and biochemical markers) include the considerable time required to conduct the experiments and the production of low-resolution and inaccurate results. Wang et al. [34] characterized nine wheat-P. huashanica chromosome addition lines, with individual alien chromosomes identified according to Giemsa C-banding, but the distributed bands were unclear, which was detrimental for identifying chromosomes. By combining a GISH, Du et al. [67] identified a complete set of 1Ns-7Ns chromosomal addition lines using EST-SSR and EST-STS markers; however, the process was laborious and time-consuming. Biochemical markers, including HMW-GS, LMW-GS, and gliadin, are useful, but only for tracing the 1Ns and 6Ns chromosomes of P. huashanica [6]. FISH analyses involving suitable probes for identifying alien chromosomes are preferred by researchers because they are relatively simple to perform and inexpensive. For example, Du et al. [68] quickly and easily clarified seven CS-Th. bessarabicum alien chromosome introgressions by completing a FISH using Th. bessarabicum-specific oligo pools. Furthermore, 17 wheat-A. cristatum introgression lines containing different P-genome chromosomes were efficiently identified by using the isolated P-genome-specific DNA sequences for a FISH [69]. Li et al. [70] rapidly determined the chromosomal composition of eight wheat-tetraploid Th. elongatum hybrid derivatives using the pSc119.2, pTa535, pTa71, and pTa713 probe combination and suggested that the E-genome chromosomes of the tetraploid Th. elongatum can be quickly and accurately identified via FISH karyotyping in the wheat background. In our study, we efficiently characterized six wheat-P. huashanica chromosome addition lines in a FISH using pSc200, pTa71A-2, and Oligo-44. Our findings indicate that the P. huashanica Ns-genome chromosomes in the wheat background can be rapidly and precisely identified by a FISH using specific probes. These lines containing monosomic or diverse alien chromosomes have been selfed to obtain stable disomic chromosome addition lines. In future investigations, we will evaluate the agronomic performance and disease resistance of these lines and assess their utility as genetic resources relevant for wheat breeding.

Conclusions
We constructed a FISH karyotype of P. huashanica using oligonucleotide probes pSc200, pTa71A-2 and Oligo-44. The combination of these probes helped distinguish all the Ns-genome chromosomes of P. huashanica (except for 4Ns) from those of wheat, diploid wheat relatives and most Ns-genome-containing species, providing an alternative for sequential GISH and FISH to detected P. huashanica chromosomes in the wheat background directly. Additionally, six wheat-P. huashanica addition lines were characterized and will be evaluated for agronomic performance and disease resistance. The FISH karyotype established here lays a solid foundation for the efficient identification of P. huashanica chromosomes in wheat genetic improvement programs.